AppliCations
No.15
AppliFect – ­
New Transfection Reagents
The term transfection describes the introduction of foreign genetic material
into eukaryotic cells. Temporary introduction of DNA or RNA into the host cell is
referred to as transient transfection, while the permanent integration into the
genome is called stable transfection. Today, cationic lipid-mediated transfection
is the most widely used method for gene transfer into eukaryotic cells in life
science ­laboratory. AppliChem offers specialized reagents for all cell types and
applications.
Keywords
lipoplex
lipofection
gene transfer
recombinant expression
gene silencing
Introduction
Today physical, biological and chemical methods are applied to introduce genetic material into the cells.
1. Physical methods such as electroporation, microinjection or bioballistics (“gene gun”) require special instruments. The
application of such methods is often restricted to special cell lines or tissues. For instance, in majority of the cases the gene
gun is used for plant cells, microinjection is a common tool to introduce RNA or DNA into C. elegans or Xenopus oocytes.
2. Biological systems for nucleic acid delivery employ viruses and protoplasts from bacteria. Viral transfection methods
­employ genetically modified viruses that are no longer pathogenic. Application of virus-mediated transfection is significantly limited by viral-related immunogenicity and the size limitation of the transgene. Both biological systems are highly
evolved methods that are not easy to use for general applications.
3. The classical chemical method is calcium phosphate precipitation. This procedure involves co-precipitation of DNA
­associated with calcium phosphate crystals that are taken up by the host cells, unfortunately also resulting in noxious effects
to most of the cells. As a result, the transfection efficiency is low, typically approx. 20 %. DEAE-Dextran is another commonly
used chemical agent for transfection. Both of the methods work well for most permanent cell lines and may be appropriate
if sufficient cell material is available to compensate for the typically low transfection rates.
The most widely applied transfection method is the chemical method of using cationic lipids, also called lipofection. The
method is easy to use and does not require any additional lab instruments. State of the art lipid formulations have only low
cytotoxic effects and often transfection rates of more than 80 % are achieved.
Transfection with cationic lipids
In aqueous solutions, cationic lipids and co-lipids form vesicles with a lipid bilayer,
known as liposomes. When liposomes encounter nucleic acids they re-form into
nucleic acid:lipid complexes called lipoplexes.
The postulated mechanism of DNA transfection involves four steps (Fig. 1).
1. Cationic lipids spontaneously form a so called lipoplex with negatively
charged DNA.
2. Eukaryotic cells take up the lipoplex via endocytosis.
3. Inside the cell the endosomal structure is destroyed by increasing the osmotic
pressure created by the lipids buffering action within the endosomes. Lipids of
the lipoplex fuse with the endosomal membrane and DNA is released into the
cytosol.
1. lipoplex
DNA
cationic lipid
4. The barrier of the nucleus is permeable to foreign DNA only during cell division
(Mitosis). A high rate of cell division thus ensures successful transfection and
good gene expression.
The optimized formulation of AppliChem‘s AppliFect solutions positively affects
crucial steps of the transfection mechanism: (i) in the formation of the lipoplex
and (ii) in the release of DNA inside the cell. The rate of cell division is only little
affected by AppliFect due to good compatibility. As a result the transfection efficiency is maximized.
co-lipid
cell membrane
cytosol
2. endocytosis
3. release of DNA
endosome
cell nucleus
Fig. 1: The mechanism of DNA lipofection comprises
formation of a lipoplex, endocytosis, DNA release
from the endosome, and entry of foreign DNA into the
nucleus during cell division.
4. DNA entry
during mitosis
The Range of AppliChem Transfection Reagents
AppliFect (A8886)
Polycationic transfection reagent for eukaryotic cells
AppliFect contains liposomes composed of a polycationic transfection reagent and
a neutral co-lipid. The efficient uptake of DNA/RNA into eukaryotic cells is mediated by complexing of the nucleic acids with the liposomes of the transfection
reagent. The transfected liposome-bound DNA/RNA is completely released within
the cells, resulting in the maximum effect of transfected nucleic acids.
GFP
With AppliFect, highest expression levels, translation or inhibition of a gene can
be achieved. The presence of serum does not interfere with the efficiency of transfections.
Examples of successfully transfected cell lines: 293T, CHO, COS-7, CV-1, HeLa,
MDCK, NIH 3T3, Vero
transmitted light
overlay
Fig. 2: Cos-7 cells transfected with GFP using AppliFect
AppliFect LowTox (A9027)
Transfection reagent for sensitive mammalian cells
AppliFect LowTox is an improved reagent for liposome-mediated transfection of
mammalian cells. AppliFect LowTox achieves highest compatibility with many
cell lines and highest transfection efficiency, while it shows only minimal cytotoxic effects. The transfected DNA or RNA is rapidly released in the cytosol from the
lipoplex of cationic lipids and co-lipids.
GFP
Fig. 3: 293T cells transfected with GFP using AppliFect LowTox
The reagent shows no serum inhibition. AppliFect LowTox is the first choice for
transfections of sensitive cell lines.
Examples of successfully transfected cell lines: 293T, BHK, CaCo-2, CHO-K1,
COS-7, CV-1, HeLa, HepG2, J774, MDCK, NIH3T3, Vero
transmitted light
overlay
AppliFect SI (A8899)
Reagent for siRNA and miRNA transfection of mammalian cells
General Considerations for Successful Transfections
AppliFect SI is an advanced lipid formulation designed for the transfection of
mammalian cells with siRNA and miRNA. Excellent knock-down results are
achieved with even low amounts of RNA (in the picogram range). The AppliFect
SI protocol for miRNA/siRNA transfection is easy to use and does not require any
further optimization of the lipoplex composition since the optimal ratio of RNA to
reagent is established for this product.
1. The cells should be proliferating and in good condition. We recommend regularly passaged cells for transfection. Microbial contaminants (such as by mycoplasma or fungi) will negatively affect the
transfection.
2. Cell divisions support the transport of DNA into the nucleus. Therefore, DNA uptake during the exponential growth phase is critical for
optimal results. Best transfection results are achieved when cells are at
30 – 60 % of “real” confluence. (This correlates with the “apparent”
confluence of 90 –100 % as seen through the microscope).
3. The DNA/RNA to be transfected should be of highest possible purity.
For example, endotoxins significantly reduce the transfection efficiency.
4. Adsorption of DNA/RNA to the reaction tube surface can cause a decrease in transfection efficiency. Polypropylene reaction tubes are the
best plastics for transfection procedures. Polypropylene has a low tendency to bind transfection reagent or genetic material in comparison
to glass or polyethylene.
AppliFect Insect (A9002)
Reagent for the transfection of insect cells
AppliFect Insect is designed for optimum liposome-mediated transfection of insect
cells. It is an aqueous formulation of positively charged lipids. Transfection rates
achieved with AppliFect Insect are significantly higher in comparison to protocols
using e.g. calcium phosphate or DEAE-Dextran. In addition, cytotoxic effects of
AppliFect are considerably lower.
Stable Transfections
In contrast to transient transfections, cells are seeded at lower cell density.
At the day of transfection cells should be less than 50 % confluent.
After transfection, cell culture medium is replaced with an appropriate
selection medium (including antibiotics).
Overview of AppliChem‘s Transfection solutions
Description
AppliFect
AppliFect LowTox
AppliFect SI
AppliFect Insect
Product code
A8886
A9027
A8899
A9002
Applications
plasmid DNA transfection,
transient or stable transfection
plasmid DNA transfection,
co-transfection of siRNA
and plasmid DNA,
transient or stable
transfection
transfection of siRNA or
miRNA for gene silencing
transfection of DNA or RNA
(suitable for Baculovirus
expression vector system,
BEVS)
Cell types
eukaryotic cells,
suspension and
adherent cells
(sensitive) eukaryotic cells,
suspension and adherent cells
mammalian cells
insect cells
(e.g. Ag55, Anso, As43, Bm5,
SF21, Mos-20, S2, SF9)
Technology
polycationic lipid reagent
formulation of cationic lipids
and co-lipids
formulation of cationic lipids
and co-lipids
cationic lipids
Features
inexpensive, economic
suitable for a wide range of
cells and applications,
minimal cytotoxicity
high transfection efficiency
low cytotoxicity
low (picogram) amount of RNA
sufficient for full knock-down
no serum inhibition
easy scale-up for production
of recombinant proteins
Number
of transfections/1ml
ca. 500
200
ca. 500
200
ca. 1000
250
ca. 100
50
Pack sizes available
A8886,00002
A8886,0001
24-well
6-well
200 µl
1ml
A9027,0001
24-well
6-well
1ml
A8899,00002
A8899,0001
96-well
24-well
6-well
60 mm dish
200 µl A9002,00005
1ml A9002,0001
AppliChem GmbH | Ottoweg 4 | D-64291 Darmstadt | Phone +49 6151 93 57-0 | Fax +49 6151 93 57-11 |
eMail [email protected] | Internet www.applichem.com
500 µl
1ml
A131,E;201104
4t Matthes + Traut · Darmstadt